Capillary electrophoresis is a technique for analyzing and/or purifying a wide variety of biochemical substances or analytes such as proteins, nucleic acids, carbohydrates, hormones and vitamins. In particular, electrophoresis is an extremely efficacious and powerful means for the identification and/or separation of analytes based upon ultra-small volume samples. In general, electrophoresis is a phenomenon that involves the migration of charged particles or analytes through a conducting liquid solution under the influence of an applied voltage.
The basic capillary electrophoretic apparatus consists of a capillary column having the ends thereof positioned in reservoirs containing electrodes. A conducting liquid or buffer solution disposed in the reservoirs and the capillary column comprises the electrophoretic conductive circuit.
An analyte is injected into the appropriate end of the capillary column and a voltage applied across the electrodes. The applied voltage causes the analyte to migrate electrophoretically through the capillary column past a prepositioned on-column detection device to generate an electropherogram, a graphical representation of the analyte.
Electrophoresis may be conducted in "open" or "gel" capillary columns. Open capillary electrophoresis can be conducted either with or without electroosmosis which involves bulk solvent migration under the influence of the applied voltage as a result of the charged condition of the inner wall of the capillary column. Gel capillary electrophoresis, in which the interior channel of the capillary column is filled with a suitable gel, provides the potential for different modes of separation based upon size of the analytes.
In either open or gel capillary electrophoresis, however, the applied voltage is a primary factor affecting the migration of the analyte. Therefore, the term electromigration as used herein encompasses either or both forms of voltage induced analyte movement.
An effective high performance capillary electrophoretic system provides high resolution, high sensitivity, short run times, on-line monitoring or detection of the analyte, and reproducible performance. One practical way of enhancing the performance of a capillary electrophoretic apparatus is by the application of high applied voltages. Another is to utilize shortened capillary columns. Both of these means of enhancing the effectiveness of the electrophoretic apparatus, however, have heretofore been limited due to the Joule heat generated in the capillary column during the electrokinetic separation operation which adversely affects electrophoretic separations.
The applied voltage causes a current flow in the buffer solution of the electrophoretic apparatus that is generally defined by Ohm's Law. The current flow through the capillary column generates Joule heat or thermal energy in the capillary column. Increasing the applied voltage increases the current flow and the resulting increased power raises the amount of Joule heat generated, which is generally an adverse condition. Similarly, shortening the length of the capillary column decreases the capillary column resistance, thereby causing an increase in current flow for a given applied voltage with the concomitant increase in Joule heating.
Column temperature influences most of the important physical and chemical parameters involved in high performance capillary electrophoresis. In particular, column temperature directly affects electrophoretic separation since there is a variation in mobility of about 2%/.degree.C. For any given electrophoretic separation, there is generally a preferred column temperature for optimal separation conditions. A column temperature deviation of only 1.degree. C. may affect the migration rate, thereby adversely impacting on separation reproducibility. Other adverse effects that may result from column temperature deviations include reduced separation efficiency, sample decomposition, and the inability to maintain the desired chemical equilibria.
Joule heat generated within the capillary column, if not efficiently controlled and/or dissipated to the ambient environment, causes a temperature buildup within the column. The temperature buildup detrimentally affects the electrophoretic separation by inducing variations in column resistance, which affects current flow, and concomitantly Joule heating, in the column FIG. 18 illustrates the variations in column current with time, with a constant applied voltage, for various methods of cooling a capillary electrophoretic column.
Curves 1 and 2, respectively, illustrate column cooling by natural convection and forced air convection with a fan. An examination of these curves reveals a noticeable variation in column current with time. Point A indicates the effect of the operation of an air conditioner in the laboratory, and shows that both natural convection and forced air cooling are susceptible to changes in the laboratory environment. Curve 3 illustrates cooling of the capillary column by means of a solid state cooling device according to the present invention. The solid state cooling device provides: (1) isolation of capillary column from ambient environmental conditions and (2) precise control of the temperature of the capillary column. FIG. 18 also graphically illustrates the fact that, although each electrophoretic separation was conducted under the same operating conditions, there was a significant deviation in column current flow, and hence column operating temperature (57.degree. C., 33.degree. C., 24.degree. C., respectively, based upon the corresponding column current flow and the mobility rate of 2%/.degree.C.), among the electrophoretic operations due to the different methods of cooling.
An optimized high performance capillary electrophoretic apparatus provides statistically reproducible results for equivalent analytes, with minimum band broadening of the output. Preferably, the apparatus is operated at high applied voltages to provide high speed, efficiency and resolution of separations. An optimized high performance capillary electrophoretic apparatus must include a temperature control system that maximizes reproducibility of column resistance and minimizes any detrimental thermal effects on separation. The temperature control system, in addition to effectively maintaining a constant column temperature, should have the capability to vary the column temperature depending upon the particular application.
For example, it may be important to vary column temperature to manipulate chemical equilibria such as metal chelation and micelle partitioning. Electrophoretic separations below ambient temperature have been shown to be useful in minimizing proteolysis or sample decomposition. Electrophoretic separation of oligonucleotides, in contrast, have been improved by injecting at 60.degree. C. where the species adopts a random coil configuration.
Prior art attempts to cool the capillary electrophoretic apparatus have involved natural and forced convection cooling. Due to disadvantages associated with many cooling solvents, e.g., low cooling capacity, flamability, toxicity, and/or high costs, water has generally been used as the cooling element in prior art electrophoretic convection cooling systems. In additional to being high capacity devices, requiring two to four liters of water, water-cooled devices suffer a marked degradation in cooling performance, about 20 to 40 percent, at temperatures approaching four degrees centigrade. And while water-cooled devices provide an improvement over air-convection devices in controlling column temperature, water-cooled devices are severely limited in capability to rapidly vary the column temperature for different applications, and a relatively expensive. In addition, a water coolant has a sufficient degree of electrical conductivity to interfere with the electrophoretic separation process.
Another limiting aspect of prior art capillary electrophoretic apparatus was due to the fact that the structural configuration of temperature regulating systems severely hindered temperature control in the detection zone. A lack of temperature control can lead to non-reproducible results in migration rates and separation. Likewise, for accurate collection of a given species in micropreparative applications, the column length between the detection zone and the collection point should be minimized, and this column length should have similar temperature characteristics to the separation region prior to the detection zone in order to predict accurately when a peak leaves the column.
Moreover, reproducible results in signal-to-noise ratio were difficult to achieve in prior art capillary electrophoretic apparatus due to the cumbersome and time consuming effort required to properly align and lock the capillary column with respect to the prepositioned detection device. Improper alignment of or failure to lock the capillary column in a predetermined position leads to the generation of variable noise due to vibration effects, which can lead to poor detection limits.